We have developed a versatile technique for ultra-high-precision laser machining that uses tightly focused femtosecond laser pulses to ablate sharply defined nanometer-scale regions in materials. With this technology it is possible to selectively ablate regions even smaller than 20 nm across. This milestone achievement holds great promise for a wide range of applications, perhaps none more exciting than as a tool to highly selectively destroy intracellular structures. The ability to create structural "knockouts" in which intracellular components are selectively destroyed holds enormous promise for elucidating structure-function relationships, just as molecular-genetic knockouts have been crucial to understanding the function of genes and the proteins they encode. Toward fulfilling this potential, this proposal has dual goals of demonstrating the utility of this approach to the broader biological community, and addressing fundamental questions concerning cell division. To these ends we propose to apply structural knockout technology to study the biomechanics of the cytoskeleton and mitosis. These experiments will characterize: 1) the mechanical and force generating properties that allow chromosomes to bind and move along microtubules, 2) the antimitotic activity of the chemotherapy drug taxol, and 3) the role of centrioles in establishing mitotic architecture. The ultimate limits of structural knockout technology will also be investigated to explore the potential for intriguing future applications such as targeted disruption of single molecules or genes.